Stem cell gene targeting

ABSTRACT

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Application Ser. No. 60/881,226filed Jan. 19, 2007, the disclosure of which is incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with government support under HL80627 andP20GM075019 awarded by the National Institutes of Health. The UnitedStates government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The derivation of human embryonic stem cells (hESCs) has opened newavenues for studies on human development and it also provides apotential source of cells for replacement therapy. For example, theability to genetically alter hESCs offers unique opportunities to studythe mechanisms regulating lineage commitment during human development,establish new approaches to identify and screen drugs, and develop invitro models of human disease. The feasibility of this approach isdependent on the identification of a locus in the genome that is easy toaccess through targeting and one that will be permissive to expressionof the introduced genetic material in the undifferentiated ES cells aswell as in a broad range of differentiated cell types generated fromthese cells. A general review of this approach is provided in Yates etal., Gene Therapy (2006) vol. 13: 1431-1439.

Previous studies aimed at expressing genes in hESCs and derivativelineages have used either lentiviral vectors or transgenes thatintegrate randomly into the genome. These approaches are problematic fora number of reasons, e.g., a randomly integrated vector can activate orsuppress expression of endogenous genes through insertional mutagenesis,the vectors are often present in multiple copies, and their expressionis subject to silencing.

Homologous recombination in mouse embryonic stem cells has been used toproduce mice carrying a single copy of the transgene integrated into apredetermined site of the genome (see e.g., Shaw-White et al.,Transgenic Res.; (1):1-13 (1993); Bronson et al., Proc. Natl. Acad. Sci.USA, 93(17:9067-72 (1996); Hatada et al., J. Biol., Chem., 274(2):948-55(1999); Tang et al., Genesis, 32(3):199-202 (2002)). In these studies,the ubiquitous Hprt locus was used with limited and unpredictablesuccess. It would be desirable to define an autosomal locus that allowsstrong and predictable expression of transgenes inserted throughhomologous recombination, but is difficult to identify chromosomal locithat fulfill these criteria. Exogenous transgenes may not harbor all ofthe sequences necessary and sufficient for proper regulation oftranscription and may therefore be influenced by cis-regulatory elementsnear the site of insertion.

In the mouse, a locus known as Rosa26 locus meets these criteria becauseit is expressed in ES cells and many derivative tissues both in vitroand in vivo and new genetic material can be easily introduced into itthrough homologous recombination. WO 99/53017 describes a process formaking transgenic animals that ubiquitously express a heterologous gene,wherein the heterologous gene is under the control of a ubiquitouslyexpressed endogenous promoter, e.g., that of the mouse Rosa26 locus. R.Dacquin et al., Dev. Dynamics 224:245-251 (2002) and K. A. Moses et al.,Genesis 31:176-180 (2001) utilize the transgenic mouse strain R26Robtained according to WO 99/53017 for the expression of heterologousgenes. WO 02/098217 describes a method of targeting promoter-lessselection cassettes into transcriptionally active loci, such as theRosa26 locus. WO 03/020743 describes the expression of transgenes invivo by targeting protected transgene cassettes into predetermined loci(e.g. the Rosa26 locus), such that the introduced tissue specificexogenous promoter has at least some tissue specific activity.

US 2006/0205077 describes a method for targeted transgenesis using themRosa26 locus. U.S. Pat. No. 6,461,864 also describes the use of themRosa26 locus in the production of genetically engineered non-humananimals that express a heterologous DNA segment.

SUMMARY OF THE INVENTION

The present invention is based on the identification of the human Rosa26(hRosa26) locus, which is capable of preserving the activity ofheterologous promoters inserted through homologous recombination at thelocus. Human Rosa26 is therefore useful for the efficient generation oftransgenic animals, tissues and cell populations with a predictabletransgene expression pattern.

Therefore, the present invention provides a method for generating atransgenic eukaryotic cell population having a modified human Rosa26locus, which method comprises introducing a functional DNA sequence intothe human Rosa26 locus of starting eukaryotic cells. In one embodiment,the functional DNA sequence is a gene expression cassette comprising agene of interest operatively linked to a heterologous promoter;alternatively, the functional DNA sequence is a gene expression cassettecomprising a gene of interest, wherein the DNA sequence becomesintegrated into the locus by homologous recombination, thereby insertingthe DNA sequence into the locus such that expression of the DNA sequenceis under the control of the endogenous hRosa26 promoter.

In the method of the present invention, the functional DNA sequence isintroduced into the eukaryotic cells by homologous recombination with atargeting vector comprising the functional DNA sequence flanked by DNAsequences homologous to the human Rosa26 locus. The eukaryotic cells areselected from the group consisting of primary cells and immortalizedcells, and in a particular embodiment the eukaryotic cells are humanembryonic stem (ES) cells.

The gene of interest may be any DNA sequence. A non-limiting list ofgenes that may be used in the method of the present invention includesrecombinases, reporter genes, receptors, signaling molecules,transcription factors, pharmaceutically active proteins and peptides,drug target candidates, disease causing gene products and toxins, andmutations and combinations thereof.

In one embodiment, the functional DNA sequence is a gene expressioncassette comprising a gene of interest operatively linked to aheterologous promoter, wherein the promoter is selected from the groupconsisting of a constitutive ubiquitous promoter, a constitutive tissuespecific promoter, an inducible ubiquitous promoter and an inducibletissue specific promoter. A non-limiting list of suitable promotersincludes CAGGS, hCMV, PGK, FABP, Lck, CamKII, CD19, Keratin, Albumin,aP2, Insulin, MCK, MyHC, WAP, Col2A, Mx, tet and Trex promoter.

The functional DNA sequence or gene expression cassette used in themethod of the present invention further comprises one or more additionalfunctional sequences selected from the group consisting of marker genes,one or more recombinase recognition sites which may be the same ordifferent, poly A signal, introns, and combinations thereof. Forexample, the expression cassette comprises one or more functionalsequences selected from the group consisting of a viral splice acceptor,a loxP-flanked promoterless neomycin resistance gene, an inverted RFPvariant, mutant loxP2272 sites, and combinations thereof. In aparticular embodiment, the expression cassette comprises the followingelements in sequential order: (a) a viral splice acceptor, (b) aloxP-flanked promoterless neomycin resistance gene, and (c) an invertedRFP variant (tdRFP), wherein said inverted RFP variant is flanked bymutant loxP2272 sites. Specifically, the expression cassette comprises aDNA sequence coding for a Cre recombinase and said loxP and mutantloxP2272 sites are positioned such that following expression of Crerecombinase, the neomycin resistance cassette is removed and the tdRFPinverted, placing it under control of the endogenous hROSA26 promoter.

Still further, the targeting vector used in the invention furthercomprises functional sequences selected from the group consisting oftags for protein detection, enhancers, selection markers, andcombinations thereof.

The transgenic eukaryotic cells are derived from human and the DNAsequences homologous to the human Rosa26 locus are derived from the 5′and 3′ flanking arm of the human Rosa26 locus. In one embodiment, thetargeting vector comprises a functional DNA sequence flanked by DNAsequence homologous with a human Rosa26 locus.

The invention also comprises a eukaryotic cell population comprising amodified human Rosa26 locus.

The invention additionally provides an isolated DNA sequencesubstantially homologous to a nucleotide sequence located betweennucleotide positions 9′415′082 and 9′414′043 on chromosome 3.Alternatively, the invention provides an isolated DNA sequencesubstantially homologous to SEQ ID NO:2. Still further, the inventionprovides an isolated DNA sequence that hybridizes under stringentconditions to the nucleic acid sequence of SEQ ID NO:2.

Also provided is a transgenic non-human animal comprising a modifiedhuman Rosa26 locus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a , FIG. 1b , FIG. 1c , FIG. 1d , FIG. 1e and FIG. 1f show theidentification, expression and targeting of the hRosa26 locus. FIG. 1ashows the expression of hRosa26 in different adult tissues and in 3 hESClines (HI, HES2, HES3). Expression of ExonIII was detected usingquantitative PCR. Data is represented as the average expression from 2to 4 individual RT/qPCR reactions. FIG. 1b shows the expression ofExonII and ExonIII evaluated using RT-PCR; +/−RT indicates the presenceof absence of reverse transcriptase. This PCR amplifies a 260 bp productspanning the putative intron. FIG. 1c is a schematic representation ofthe targeting vector and a segment of the newly identified human Rosa26locus. Grey triangles denote wild-type loxP and white triangles mutantloxP2272 sites (SA=splice acceptor). The box with vertical barsindicates the region of highest sequence homology (>85%) between mouseand human. Putative exons 1 and 2 were mapped according to the positionsof electronically identified ESTs/transcripts. FIG. 1d shows the humanRosa26 locus after gene targeting and cremediated activation of tdRFP.FIG. 1e is a Southern blot of parental HES2 and targeted hRosa26 celllines before (WT/KI) and after Cre mediated tdRFP inversion (WT/KItdRFP). WT=wild type, KI=knock-in. FIG. 1f shows hRosa26 genomic DNAhybridized with a tdRFP specific probe identifying a single integrationevent.

FIG. 2a , FIG. 2b , FIG. 2c , FIG. 2d , FIG. 2e , FIG. 2f , FIG. 2g ,FIG. 2h and FIG. 2i show the morphology and differentiation of hRosa26ES cells. FIG. 2a shows the morphology of the targeted hRosa26 ES cellsgrown on mouse embryonic feeder cells using light microscopy. FIG. 2bshows the expression of tdRFP in hRosa26 ES cells revealed byfluorescence microscopy. FIG. 2c shows the alkaline phosphataseexpression in the targeted in hRosa26 ES cells grown on Matrigel for 4passages. FIG. 2d shows five-day-old EBs derived from hRosa26 ES cells;phase contrast. FIG. 2e shows the expression of tdRFP in hRosa26 EBsrevealed by fluorescence microscopy and FIG. 2f shows the flowcytometric analysis of the targeted hRosa26 cells (shaded and withouthatching), the parental HES2 cells (hatched and shaded) and cells fromday 17 hRosa26 EBs (bold line). Teratoma derived from hRosa26 ES cellsshown under bright (FIG. 2g ) and epifluorescent light (FIG. 2h ). Theinserts show single cells from a different hRosa26 subclone (phasecontrast and fluorescence microscopy, FIG. 2g and FIG. 2h ,respectively). FIG. 2i shows the flow cytometric analysis showing tdRFPexpression in a large majority (>96%) of the teratoma cells. Live cells(>60%) were gated based on forward and side scatter parameters.

FIG. 3a , FIG. 3b , FIG. 3c , FIG. 3d and FIG. 3e show multilineagedifferentiation of hRosa26 targeted hESCs in vitro. FIG. 3a showsdeveloping neurons expressing β-Tubulin III (green) and tdRFP (red).Total population is visualized by nuclear DAPI staining (blue). Insert:overlay demonstrating co-expression of tdRFP and β-Tubulin III. FIG. 3bshows tdRFP expression in AFP cells generated from hRosa26 (upper panel)and wild type (HES2) cells (lower panel). The first column represents anoverlay of an IgG control (green), tdRFP and DAPI. The second, third andfourth column show individual channels for AFP staining (green), tdRFP(red) and DAPI (blue), respectively. FIG. 3c shows myeloid (M) anderythroid (E) hematopoietic colonies grown from day 18 EBs generatedfrom the hRosa26 cells. The insert shows tdRFP expression in both typesof colonies. Exposure with the GFP filter is included to control forauto fluorescence (EGFP). FIG. 3d shows flow cytometric analysisdemonstrating CD45 and tdRFP expression at days 14 and 21 in EB-derivedcells generated from hRosa26 hESCs (red) or wild type hESCs (blue). FIG.3e shows the expression of tdRFP in human chorionic gonadotropinpositive cells generated from hRosa26 cells (left, center left andcenter right) and HES2 parental cells (right). The hESC weredifferentiated in serum free media with high concentrations of humanBMP4 (100 ng/ml) to induce trophectoderm differentiation. After 14 dayscells were stained with an antibody specific for human chorionicgonadotropin subunit B (hCG) or an isotype control (center right, IgG).

FIG. 4a and FIG. 4b show the alignment of the mouse and human Rosa26sequences and multiple alignment plot of selected human ESTs. FIG. 4ashows the alignment of the mouse and human Rosa26 sequences with thehighest degree of homology (>85%; box with vertical bars in FIG. 1c ;the mouse sequence depicted in FIG. 4a is SEQ ID NO: 1 and the humansequence depicted in FIG. 4a is SEQ ID NO: 2). The top arrow denotes the5′ start of the mouse Rosa26 transcript 1, the bottom arrow indicatesthe start of the most 5′ human transcript found in Ensembl database(GenBank: CR624523). The human sequence shown is located betweennucleotide positions 9′415′082 and 9′414′043 on chromosome 3 (EnsemblHuman Blast View v37). FIG. 4b shows the multiple alignment plot ofselected human ESTs showing local similarities to the genomic sequenceof the putative hRosa26 locus. Areas of significant similarities (≧60%)are boxed. Each EST is labeled with its GenBank accession number and thetissue source. Predicted exons are indicated as black bars on a genomicDNA representation and numbered using Roman numerals. In the mouse,Rosa26 overlaps with the ThumpD3 gene which is positioned in the reverseorientation downstream of the Rosa26 transcription unit. To highlightthe high degree of synteny between this human chromosomal region and themouse Rosa26 locus, exon structure of the human THUMPD3 is alsorepresented as gray bars.

FIG. 5a and FIG. 5b show single integration in the hRosa26 hESC. FIG. 5ais a schematic drawing of the hRosa26 locus after Cre mediated tdRFPactivation. EcoR1=EcoR1 restriction enzyme, ProbeRFP=1.4 kb tdRFPinternal Southern blot probe. FIG. 5b shows hRosa26 genomic DNA wasdigested with EcoRI and hybridized with a tdRFP specific probe (same gelas in FIG. 1f ). 1 kb=1 kb plus DNA ladder (Invitrogen), EtBr=ethidiumbromide.

FIG. 6a and FIG. 6b show the identification and characterization of asecond hRosa26 clone (hRosa26.2). FIG. 6a shows hRosa26.2 and controlgenomic DNA were digested with HindIII and hybridized with a 900 bpexternal genomic probe revealing the 6 kb wild-type (WT) and 3.5 kbknock-in (KI) allele. FIG. 6b shows hRosa26.2 and HES2 parental cellswere differentiated under serum free conditions to mesoderm andhematopoietic cells. After 17 days of differentiation EB-derived cellswere analyzed for the expression of tdRFP and CD45.

FIG. 7a , FIG. 7b , FIG. 7c and FIG. 7d show a histological analysis ofhRosa26-derived teratoma ES cells injected into the hindleg muscle ofNOD/SCID mice and resulting teratomas were analyzed 9 weeks later.Hematoxylin/eosin (H&E) stained paraffin sections from these teratomasdemonstrate contribution to all 3 germ 20 layers: FIG. 7a . cartilage(*). FIG. 7b . striated cardiac muscle with centrally located nuclei(arrow). FIG. 7c . ciliated mucosal tissue (*) with secretory Gobletcells (arrow). FIG. 7d . neural rosettes (arrow).

FIG. 8a , FIG. 8b , FIG. 8c , FIG. 8d and FIG. 8e show RMCE in hRosa26ES cells. FIG. 8a is a schematic representation of the hRosa26 genomiclocus before targeting; FIG. 8b shows the locus after targeting and Cremediated tdRFP inversion; and FIG. 8c shows the locus following exchangeof the tdRFP to puroTK with RMCE. Filled triangles denotes the wild-typeloxP site and the open triangles the mutant loxP2272 site. SA=spliceacceptor. FIG. 8d is a Southern blot of genomic DNA (digested with XbaI)hybridized with a puro specific probe revealing a single integration at6.1 kb. FIG. 8e is a southern blot analysis of hRosa26 clones before andafter RMCE. Genomic DNA was digested with HindIII and subsequentlyhybridized with a hRosa26 external probe (same probe as in FIG. 1e ).

FIG. 9a and FIG. 9b show inducible expression from the human Rosa26locus. Using RMCE, an inducible expression cassette was introduced intothe human Rosa26 locus. This vector contains both thetetracycline-controlled transactivator as well as a tetracyclineresponse element. In addition a transgene can be introduced and isexpressed upon the removal of doxycycline. Expression of the transgenecan be monitored by the expression of the Venus fluorescent proteinexpressed from an internal ribosomal entry site (IRES). As shown in FIG.9a , in the presence of doxycycline (+Dox) no Venus protein can bedetected by flow cytometry, however upon removal of dox high levels ofthe fluorescent reporter can be detected (FIG. 9b ).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the human homologue of the mouse Rosa26locus and methods for site-specific integration of a transgene into thehuman Rosa26 locus. Therefore, the invention provides an isolated DNAsequence substantially homologous to a nucleotide sequence locatedbetween nucleotide positions 9′415′082 and 9′414′043 on chromosome 3.The invention further provides an isolated DNA sequence substantiallyhomologous to SEQ ID NO: 2. And also provided is an isolated DNAsequence that hybridizes under stringent conditions to the nucleic acidsequence of SEQ ID NO:2.

The terms “substantially homologous”, “substantially corresponds to”,and “substantial identity” as used herein denotes a characteristic of anucleic acid sequence such that a nucleic acid sequence has at leastabout 90% sequence identity, and most preferably at least about 95%sequence identity as compared to a reference sequence. The percentage ofsequence identity is calculated excluding small deletions or additionswhich total less than 25 percent of the reference sequence. Thereference sequence may be a subset of a larger sequence, such as aportion of a gene or flanking sequence, or a repetitive portion of achromosome. However, the reference sequence is at least 18 nucleotideslong, typically at least about 30 nucleotides long, and preferably atleast about 50 to 100 nucleotides long.

“Substantially complementary” as used herein refers to a sequence thatis complementary to a sequence that substantially corresponds to areference sequence. In general, targeting efficiency increases with thelength of the targeting transgene portion (i.e., homology region) thatis substantially complementary to a reference sequence present in thetarget DNA (i.e., crossover target sequence). In general, targetingefficiency is optimized with the use of isogenic DNA homology clamps,although it is recognized that the presence of various recombinases mayreduce the degree of sequence identity required for efficientrecombination.

“Stringent conditions” refer to conditions under which a specific hybridis formed. Generally, stringent conditions include conditions underwhich nucleic acid molecules having high homology, for example,preferably 90% or most preferably 95%, hybridize with each other.Alternatively, stringent conditions generally include conditions wherebynucleic acid molecules hybridize with each other at a salt concentrationcorresponding to a typical washing condition of Southern hybridization,i.e., approximately 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at60° C.

Any method known in the art may be used for the site-specificintegration of a transgene using the human Rosa26 locus. Suitable, butnon-limiting examples of various methods that may be used in connectionwith the hRosa26 locus are described in Yates et al., Gene Therapy(2006) vol. 13: 1431-1439; Shaw-White et al., Transgenic Res.; (1):1-13(1993); Bronson et al., Proc. Natl. Acad. Sci. USA, 93(17:9067-72(1996); Hatada et al., J. Biol., Chem., 274(2):948-55 (1999); Tang etal., Genesis, 32(3):199-202 (2002); WO 99/53017; R. Dacquin et al., Dev.Dynamics 224:245-251 (2002); K. A. Moses et al., Genesis 31:176-180(2001); WO 03/020743; US 2006/0205077; and U.S. Pat. No. 6,461,864; thedisclosures of which are incorporated herein by reference in theirentirety.

For example, a targeting vector may be engineered for the site-specificintegration of a transgene using the hRosa26 locus by methods known inthe art. A targeting vector generally comprises a first sequencehomologous to a portion or a region of a target gene sequence, i.e., thehRosa26 locus, and a second sequence homologous to a second portion orregion of a target gene sequence, i.e., a second portion of the hRosa26locus. The targeting vector may also include a selectable markercassette that comprises a selectable marker gene. Preferably, theselectable marker cassette is positioned in between the first and thesecond sequence homologous to a region or portion of the target genesequence. The selectable marker cassette may further comprise a sequencethat initiates, directs, or mediates transcription of the selectablemarker and the targeting vector also comprises a regulator that has theability to control or regulate the expression of the selectable marker.

In one embodiment of the invention, the functional DNA sequenceintroduced into the hRosa26 locus is a gene expression cassettecomprising a gene of interest operatively linked to a heterologouspromoter. As used herein, the term “promoter”, generally refers to aregulatory region of DNA capable of initiating, directing and mediatingthe transcription of a nucleic acid sequence. Promoters may additionallycomprise recognition sequences, such as upstream or downstream promoteror enhancer elements, which may influence the transcription rate.

In one embodiment of the present invention, a promoter may be used inthe gene expression cassette (which is a heterologous promoter relativeto the hRosa26 locus) that is a ubiquitous or tissue specific promoter,either constitutive or inducible. This ubiquitous promoter is selectedfrom polymerases I, II and III dependent promoters, preferably is apolymerase II or III dependent promoter including, but not limited to, aCMV promoter, a CAGGS promoter, a snRNA promoter such as U6, a RNAse PRNA promoter such as H1, a tRNA promoter, a 7SL RNA promoter, a 5 S rRNApromoter, etc. Suitable examples of ubiquitous promoters are CAGGS,hCMV, PGK, and examples of tissue specific promoters are FABP (Saam &Gordon, J. Biol. Chem., 274:38071-38082 (1999)), Lck (Orban et al.,Proc. Natl. Acad. Sci. USA, 89:6861-5 (1992)), CamKII (Tsien et al.,Cell 87: 1317-1326 (1996)), CD19 (Rickert et al., Nucleic Acids Res.25:1317-1318 (1997)); Keratin (Li et al., Development, 128:675-88(201)), Albumin (Postic & Magnuson, Genesis, 26:149-150 (2000)),aP2(Barlow et al., Nucleic Acids Res., 25 (1997)), Insulin (Ray et al.,Int. J. Pancreatol. 25:157-63 (1999)), MCK (Bruning et al., MolecularCell 2:559-569 (1998)), MyHC (Agak et al., J. Clin. Invest., 100:169-179(1997), WAP (Utomo et al., Nat. Biotechnol. 17:1091-1096 (1999)), Col2A(Ovchinnikov et al., Genesis, 26:145-146 (2000)); examples of induciblepromoter sites are Mx (Kuhn et al. Scinence, 269: 1427-1429 (1995)), tet(Urlinger et al., Proc. Natl. Acad. Sci. USA, 97:7963-8 (2000)), Trex(Feng and Erikson, Human Gene Therapy, 10:419-27). Suitable induciblepromoters are the above-mentioned promoters containing an operatorsequence including, but not limited to, tet, Ga14, lac, etc.

Alternatively, as described hereinabove, the expression cassette maycomprise a gene of interest and other integration elements such thatwhen the DNA sequence is integrated into the locus by homologousrecombination, expression of the DNA sequence is under control of theendogenous hRosa26 promoter. For example, the expression cassette mayinclude a sequence flanked by a recombinase recognition site, e.g.,loxP, and the cassette is engineered such that following expression ofthe recombinase, the functional DNA sequence is placed under the controlof the endogenous hRosa26 promoter. In particular, the expressioncassette may include a viral splice acceptor, followed by a loxP-flankedpromoterless neomycin resistance gene, which is followed by an invertedRFP variant (a tandem-dimer RFP or tdRFP), flanked by mutant loxP2272sites. As described above, the loxP and loxP2272 sites are positionedsuch that after Cre expression, the neomycin resistance cassette isremoved and the tdRFP is inverted, thereby placing the cassette underthe control of the endogenous hRosa26 promoter.

The targeting vector, functional DNA sequence or gene expressioncassette may further comprise one or more additional sequences includingbut not limited to (selectable) marker genes (such as the neomycinphosphotransferase gene of E. coli transposon, etc.), recombinaserecognition sites (which include loxP, FRT, variants thereof, etc.),poly A signals (such as synthetic polyadenylation sites, or thepolyadenylation site of human growth hormones, etc.), splice acceptorsequences (such as a splice acceptor of adenovirus, etc.), introns, tagsfor protein detection, enhancers, selection markers, etc.

In a preferred embodiment, the targeting vector comprises a functionalDNA sequence flanked by DNA sequences homologous to the human Rosa26locus. Although the size of each flanking region is not critical and canrange from as few as 100 base pairs to as many as 100 kb, preferablyeach flanking fragment is greater than about 1 kb in length, morepreferably between about 1 and about 10 kb, and even more preferablybetween about 1 and about 5 kb. Although larger fragments may increasethe number of homologous recombination events in ES cells, largerfragments will also be more difficult to clone.

In another embodiment, the method of the invention includes homologousrecombination and the expression cassette is free of a transcriptionalstop signal 5′ to the (heterologous) promoter of the cassette (i.e. is anon-protected cassette); and/or the exogenous promoter is a ubiquitous(constitutive or inducible) promoter.

The hRosa26 locus may be used for the site-specific integration of atransgene, wherein the transgene includes a gene of interest. As usedherein, a transgene includes a gene or any DNA sequence that has beenintroduced into a targeting vector and ultimately into a different cellpopulation or organism. This non-native segment of DNA may retain itsoriginal biological properties and functions, e.g., to produce RNA orprotein, once transferred or introduced into the transgenic organism, orit may alter the normal function of the transgenic organism's geneticcode.

The gene of interest includes any gene or DNA sequence of natural orsynthetic origin. A non-limiting list of genes that may be used in themethod of the present invention is selected from the group of genesconsisting of recombinases, reporter genes, receptors, signalingmolecules, transcription factors, pharmaceutically active proteins andpeptides, drug target candidates, disease causing gene products andtoxins, and mutations and combinations thereof. The term “mutation” isunderstood to mean any changes introduced into the DNA sequence of areference gene.

In one embodiment, the ES cell is a human ES cell. ES cells may beobtained commercially or isolated from blastocysts by methods known inthe art, as described for example by U.S. Pat. No. 5,843,780; Thompsonet al. (1998) Science 282:1145-1147; U.S. Pat. No. 6,492,575; Evans etal. (1981) Nature 292:154-156; and Reubinoff et al. (2000) NatureBiotech. 18:399. The method described herein may also be used to delivera transgene to an adult, i.e. somatic, stem cell. Adult stem cellsinclude, for example, hematopoietic stem cells, bone marrow stromal stemcells, adipose derived adult stem cells, olfactory adult stem cells,neuronal stem cells, skin stem cells, and so on. Adult stem cells have asimilar ability as ES cells to give rise to many different cell types,but have the advantage that they can be harvested from an adult.

The undifferentiated ES cells are preferably maintained under conditionsthat allow maintenance of healthy colonies in an undifferentiated state.For example, human ES cells may be maintained on a feeder layer such asirradiated mouse embryonic fibroblasts in the presence of serum, or withserum replacement in the presence of bFGF, or in medium conditioned bymouse embryonic fibroblasts, or under serum free conditions using humanfeeder layers derived from, for example, human embryonic fibroblasts,fallopian tube epithelial cells or foreskin.

The method of the present invention results in site-specific integrationof the transgene at the hRosa26 locus of the ES cell genome. The EScells having the integrated transgene undergo normal embryoid body (EB)development and retain the capacity to differentiate into multiple celltypes. Expression of the transgene is maintained throughoutdifferentiation. Further, the ES cells having the integrated transgenemaintain the capacity to generate cells of multiple lineages.

Stem cells having a transgene integrated therein as made by the methodof the present invention are useful, inter alia, for generatingtransgenic non-human animals, for generating differentiated cells andtissues having a transgene integrated therein, for studyingdifferentiation of stem cells, for evaluating strategies for safe andeffective gene targeting in stem cells, and for targeted therapeuticgene transfer. Methods for generating differentiated cells from stemcells are known in the art. The model system for ES cell in vitrodifferentiation is based on the formation of three dimensionalstructures known as embryoid bodies (EBs) that contain developing cellpopulations presenting derivatives of all three germ layers and isdisclosed in the art, for example by Keller (1995) Curr.^(Q)pin. CellBiol. 7:862-869.

For example in one embodiment, prior to differentiation, ES cells areremoved from feeder cells prior to differentiation by subcloning the EScells directly onto a gelatinized culture vessel. Twenty-four to 48hours prior to the initiation of EB generation, ES cells are passagedinto IMDM-ES. Following 1-2 days culture in this medium, cells areharvested and transferred into liquid medium (IMDM, 15% FBS, glutamine,transferrin, ascorbic acid, monothioglycerol and protein free hybridomamedium II) in Petri-grade dishes. Under these conditions, ES cells areunable to adhere to the surface of the culture dish, and will generateEBs.

Culture conditions are known in the art for the differentiation to celltypes found in blood (Wiles et al. (1991) Development 111:259-67), heart(Maltsev et al. (1993) Mech. Dev. 44:41-50), muscle (Rohwedel et al.(1994) Dev. Biol. 164:87-101), blood vessels (Yamashita et al. (2000)Nature 408:92-96), brain (Bain et al. (1995) Dev. Biol. 168:342), bone(Buttery et al. (2001) Tissue Eng. 7:89-99) and reproductive system(Toyooka et al. (2003) Proc. Natl. Acad. Sci. USA 100:11457-11462).

The differentiated cells and/or tissue generated therefrom may beintroduced in an animal for therapeutic purposes. Accordingly, inanother embodiment the present invention provides an animal comprisingdifferentiated cells having a transgene integrated into the hRosa26locus thereof, or comprising a tissue generated from such cells. In oneembodiment the differentiated cell is a hemotopoietic cell, endothelialcell, cardiomyocyte, skeletal muscle cell or neuronal cell. The cells ortissues may be transplanted into the animal by methods known in the art.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated herein by referencein their entireties.

EXAMPLES

Materials and Methods:

Human ES Culture:

The human embryonic stem cell line HES 2 (ES Cell International) wasgrown on irradiated Swiss Webster or DR4 mouse embryonic feeder cells(MEF, The Jackson Laboratory) in DMEM/F12 supplemented with 20% (v/v)knockout serum replacement, 5% (v/v) MEF conditioned medium, bFGF (5 to20 ng/mL, R&D Systems), 50 U/mL penicillin, 50 μg/mL streptomycin (P/S),2 mM L-glutamine (Glut), 0.1 mM non essential amino acids (all fromInvitrogen), 0.1 mM βmercapto-ethanol (β-ME)29. MEF cells were generatedand cultured as described in. Kennedy, M. & Keller, G. M. Hematopoieticcommitment of ES cells in culture. Methods Enzymol 365, 39-59 (2003).

hRosa26 Morphology:

hRosa26 hESCs were grown on either MEF cells or Matrigel (BectonDickinson). Alkaline phosphatase staining was performed as described bythe manufacturer (VectorLabs). tdRFP expression was detected using aLEICA DM IRB inverted microscope.

Identification of Putative Transcripts from the Human Rosa26 Locus:

To isolate human ESTs showing similarities to the human Rosa26 locus,putative exons were identified by comparing published mouse Rosa26transcripts to the human ROSA26 genomic sequence. Each individual exonwas then blasted against the whole human EST database using BlastN(NCBI). ESTs showing a significant E value (E>0,0001) were globallyaligned against the putative human Rosa26 genomic sequence using theAlign Plus 5 software from Scientific & Educational Software (Penaltiesfor Mismatch: 2, Gap Opening: 4, Gap Extension: 1). For analysis byquantitative PCR (qPCR) and RT-PCR total RNA from brain, pancreas, lung,kidney, bone marrow and skeletal muscle was purchased from Clontech, orisolated from HES cell lines H1 (WiCell), HES2, HESS (both ES CellInternational) and hRosa26 using the RNeasy kit (Qiagen). Reversetranscription was used to generate cDNA (Omniscript RT, Qiagen).

Human β-ACTIN cDNA was detected using the following primers: fwd 5′ TTTGAA TGA TGA GCC TTC GTC CCC 3′ (SEQ ID NO: 3) and rv 5′ GGT CTC AAG TCAGTG TAC AGG TAA GC 3′ (SEQ ID NO: 4). hRosa26 ExonIII cDNA was detectedby qPCR using the following primer set: fwd 5′ TTA TCC GTT GCG TAA GCACAG AGA GG 3′ (SEQ ID NO: 5) and rv 5′ TTA TTC TCA CGG TGT GCA GAG GCT3′ (SEQ ID NO: 6). ExonII and ExonIII cDNA by RT-PCR was detected usingthe following primer set: fwd 5′ AGA ACT GGA AGT AAA CGA TTG AAG A 3′(SEQ ID NO: 7) and rv 5′ TTA TTC TCA CGG TGT GCA GAG GCT 3′ (SEQ ID NO:8). For qPCR analysis samples were analyzed on an ABI 7900HTthermocycler and amplification was detected using the SYBR green method(BioRad, iQ SYBR green supermix). Linearity of amplification was testedon genomic DNA samples. The amount of target, normalized to the β-ACTINreference and relative to the H1 hESC line was calculated by: 2−ΔΔCT.The resulting qPCR product was confirmed by sequencing and showedhRosa26 sequence.

Vector Design:

The targeting vector was constructed using the backbone of pHL-HH, amurine Rosa26 targeting vector8. It consisted of 5′ and 3′ arms ofhomology, a loxP/loxP2272 flanked neomycin resistance cassette, aninverted loxP/loxP2272 flanked tdRFP and a diphtheria toxin negativeselection cassette. The homology arms were amplified by long rangegenomic PCR from hESC line HES2 using PCR primers introducing therestriction sites indicated: short arm fwd 5′ ATG CGT CGA CGG CTC CTCAGA GAG CCT CGG CTA GGT AGG G 3′ (SalI) (SEQ ID NO: 9) and rv 5′ TAG GGTTAA TTA AAG ATC ACG CGA GGA GGA AAG GAG GG 3′ (PacI) (SEQ ID NO: 10),long arm fwd 5′ TAT GGC GCG CCC GTC ATC GCC TCC ATG TCG AGT CGC TT 3′(AscI) (SEQ ID NO: 11) and rv 5′ TAG CGA TAT CAA ATC AGA GAC AGA AAA GTCTTT GTC ACC 3′ (EcoRV) (SEQ ID NO: 12). Subsequently the murine SA andLA in the plasmid pHL-HH were exchanged to the human counterpart. A mockscreening plasmid was generated with an extended 5′ homoly arm which wasintroduced as a linker using the following oligonucleotides: fwd 5′ TCGACG AGA AGA GGC TGT GCT TCG GCG CTC CC 3′ (SEQ ID NO: 13) and rv 5′ TCGAGG GAG CGC CGA AGC ACA GCC TCT TCT CG 3′ (SEQ ID NO: 14). The RMCEexchange vector was created by ligating an oligonucleotide linkerharboring a loxP and mutant loxP2272 site as well as multiple cloningsite (MCS) into the SacI/KpnI sites of plasmid pBluescript II SK+(Stratagene). Linker sequence is as follows: fwd 5′ ATA ACT TCG TAT AATGTA TGC TAT ACG AAG TTA TGC TAG CTC ATG GAA CGC GTA TAA CTT CGT ATA AGGTAT CCT ATA CGA AGT TAT AGC T 3′ (SEQ ID NO: 15) and rv 5′ ATA ACT TCGTAT AGG ATA CCT TAT ACG AAG TTA TAC GCG TTC CAT GAG CTA GCA TAA CTT CGTATA GCA TAC ATT ATA CGA AGT TAT GTA C 3′ (SEQ ID NO: 16). Thepuromycin-thymidine-kinase (puroTK) cassette from pBSKpuroTK wasintroduced into the MCS using standard molecular techniques.

Plasmid Preparation and Electroporation:

Forty μg of the targeting plasmid was linearized using KpnI restrictionenzyme (NEB). One day prior to electroporation DR4 feeder cells wereplated on 10 cm tissue culture treated dishes coated with a thin layerof Matrigel. For DNA electroporation human ES cells (passage 16) werecollected by trypsinization and dissociated into single cells bytrituration. Trypsin activity was inhibited using 50% (v/v) fetal calfserum (Atlas). 5×106 cells were electroporated using a BioRad genepulser (BioRad, 250V/500 μFd14 in 500 μL HES media+300 μL PhosphateBuffered Saline (PBS) containing the linearized targeting plasmid.Following electroporation the cells were plated on DR4 MEF cells at2.5×106 cells/10 ml. Twenty-four hours later G418 (Invitrogen) was addedto the culture at 25 μg/mL. The concentration of G418 was increased to50 μg/mL at day 4 of culture. Media was changed every one to two days.The resulting colonies were screened on day 12 for correct targeting ofthe locus by polymerase chain reaction (PCR) using the following primerset: fwd 5′ GAG AAG AGG CTG TGC TTC GG 3′ (SEQ ID NO: 17) and rv 5′ AAGACC GCG AAG AGT TTG TCC 3′ (SEQ ID NO: 18) amplifying a 1.3 kb fragment.

Transient Cre Expression and Cell Sorting:

hRosa26 clone B7 was expanded and electroporated with a Cre expressingplasmid (pCRE-AC, HJF, unpublished). Following electroporation cellswere plated on Matrigel coated plates in HES medium. Forty-eight hoursafter electroporation cells were harvested using trypsin-EDTA andresuspended in cell sorting media (IMDM+10% FCS) and sorted based on theexpression of tdRFP. Two thousand tdRFP+ sorted cells were plated onembryonic feeder cells in hESC media. Individual tdRFP+ clones wereidentified, picked using a glass pipette and expanded.

Southern Blot Analysis:

hRosa26 targeting was confirmed by Southern blot analysis using a 5′external probe amplified from genomic DNA using the following primerpair: fwd 5′ GCC CAA GAA GTG AGA CAA GC 3′ (SEQ ID NO: 19), rv 5′ GACAAG GTA AGG GTC CGA CA 3′ (SEQ ID NO: 20), generating a 900 bp probe.Genomic DNA was digested using the HindIII restriction enzyme. Expectedband sizes are 6 kb for the wild type allele and 3.5 kb for the targetedallele. For the internal probe a 1.4 kb tdRFP probe derived from thetargeting vector was used that would detect one 4.1 kb band(EcoR1digest), if a single integration event has occurred. For RMCESouthern blot analysis using an internal probe genomic DNA was digestedusing the restriction enzyme Xba1 and then hybridized with a 1.6 kbpuroTK probe derived from plasmid pBSKpuroTK. This probe will detect one6.1 kb band if a single copy of the construct was integrated.

ES Differentiation:

For all differentiation experiments hESCs were maintained on irradiatedMEF cells in HES media. Prior to the onset of the experiments cells werepassaged once on Matrigel coated plates to reduce the number of feeders.All cytokines were purchased from R&D Systems.

Neuroectoderm Differentiation:

Cells were harvested by trypsinization and induced to form EBs byplating at a concentration of 50,000 cells/100 μl in 96-well low clusterplates (Corning) in the following media: 50% (v/v) DMEM-F12, 50% (v/v)Neurobasal media (both from Invitrogen) supplemented with P/S, Glut,4.5×10-4 M monothioglycerol (MTG), 0.5 mM ascorbic acid (AA), 0.1% (m/v)albumin (all from Sigma), 0.5×N2 and 0.5×B27 supplement (both fromInvitrogen) (SF-ES). At day 4 of differentiation bFGF was added at aconcentration of 20 ng/mL. At day 6 cells were transferred to gelatincoated 6 well plates and media was supplemented with epidermal growthfactor (EGF, 20 ng/mL). Medium was subsequently changed every 3-5 daysusing SF-ES medium supplemented with bFGF and EGF. In some experimentscells were grown on glass coverslips, embedded in the gelatin coatin.Cells were analyzed after 21 days in culture. Antibody staining wasperformed as follows: cells were fixed in PBS+4% Para formaldehyde (PFA,EMS), blocked with serum free protein block (DAKO) and subsequentlystained with monoclonal mouse anti β-tubulin III antibody (Tu-20,Chemicon) or appropriate isotype control Staining was revealed with adonkey anti mouse IgG FITC conjugated secondary antibody (Biosource).Images were taken using either an inverted Leica DM IRB or Leica DM RA2microscope. If indicated DAPI nuclear counterstaining was performedusing DAPI mounting media (Molecular Probes).

Endoderm Differentiation:

For endoderm differentiation, hESC colonies were dissociated to smallaggregates by sequential treatment with collagenase B (Roche) for 45minutes at 370 C and subsequent treatment with trypsin-EDTA for 3minutes at 370 C (Cellgro). Following this enzymatic dissociation, thecells were scraped from the plate and gently passed 5 times through a 2ml pipette, yielding aggregates of 5-10 cells. The aggregates wereplated in low adherence culture dishes in StemPro 34 (Invitrogen),supplemented with P/S, Glut, 0.5 mM AA and 4.5×10-4 M MTG. After 3 daysEBs were harvested and cultured in 75% (v/v) IMDM, 25% (v/v) Ham's F12(Invitrogen) supplemented with P/S, Glutamine, 4.5×10-4 M MTG, 0.5 mMAA, 0.5×N2 supplement, 0.5×B27 supplement without Vitamin A and 0.1%albumin (SF-D). Activin A was added at 100 ng/mL. On day 8 EBs wereplated on gelatin coated 6 well plates in SF-D, supplemented with humanVEGF 5 ng/mL, bFGF 20 ng/mL and human BMP4 50 ng/mL. After a total of 16days in culture, cells were analyzed by immunohistochemistry for theexpression of AFP. To detect AFP, cells were fixed in PBS+4% PFA,blocked with serum free protein block and subsequently stained withrabbit anti-mouse polyclonal AFP antibody (Neomarkers) or theappropriate isotype control. Staining was revealed with a donkey antirabbit Cy2 conjugated secondary antibody (Jackson Immuno Research).Images were taken using an inverted Leica DM IRB microscope. DAPInuclear counterstaining was performed where indicated (MolecularProbes).

Mesoderm Differentiation:

Small clumps of undifferentiated hESCs were generated as describedabove. Clumps were plated in low adherence culture dishes in StemPro 34,supplemented with P/S, Glut, AA and MTG and BMP4 10 ng/mL. After one daybFGF was added at 10 ng/mL. On day 4 media was changed to fresh mediacontaining the following cytokines: VEGF 10 ng/mL, Stem Cell Factor(SCF) 100 ng/mL, erythropoietin (EPO) 4000 U/mL, IL6 10 ng/mL, IL3 20ng/mL and IL11 5 ng/mL. Three days later the EBs were transferred toIMDM supplemented with plasma derived serum (PDS, Animal Technologies)5%, protein free hybridoma medium (PFHM-II) 5% (v/v) (Invitrogen), Glut,0.5 mM AA, 3×10-4 M MTG, VEGF 10 ng/mL, SCF 100 ng/mL, EPO 4000 U/mL,IL6 10 ng/mL, IL3 20 ng/mL and IL11 5 ng/mL, TPO 40 ng/mL and IGF-1 25ng/mL. Cells were either grown in this media for the remainder of theexperiment by changing media every 5-7 days (liquid expansion) or wereswitched to a methylcellulose based semi-solid media to reveal theircolony forming potential.

Methylcellulose Assay:

At day 16 to 18 of mesoderm differentiation, EBs were separated fromfloating cells by gravity settlement. Both populations were harvestedindividually to expose only the EB fraction to collagenase type I for 60minutes, followed by trypsinization for 3 to 10 minutes. After vigorousvortexing cells from the EB fraction were passed 5 times through a 21Gneedle. Both fraction were then pooled and passed through a cellstrainer. Cells were counted and were either analyzed by FACS or platedat a concentration of 50,000 cells/ml of semi-solid media as follows: 1%(m/v) methylcellulose (Fluka), supplemented with P/S, Glut, 15% (v/v)PDS, 5% (v/v) PFHM-II, VEGF 10 ng/mL, SCF 100 ng/mL, EPO 4000 U/mL,GM-CSF 2 ng/mL, IL6 10 ng/mL, IL3 20 ng/mL and IL11 5 ng/mL, TPO 40ng/mL, IGF-1 25 ng/mL and IMDM to 100%. Colonies were analyzed at day 10of methylcellulose culture.

Teratoma Formation:

ES cells were dissociated into small aggregates by treatment withcollagenase type I for 45 minutes. Following this enzymaticdissociation, the cells were scraped from the plate and gently passed 5times through a 2 ml pipette, yielding aggregates of 15 to 20 cells. Theequivalent of 10,000 cells was resuspended in a 1:1 mixture of IMDM andMatrigel and injected into the hindleg muscle of NOD/SCID mice (TheJackson laboratory). Mice were sacrificed 8-10 weeks later and theteratomas were harvested. Red fluorescent pictures were taken using astereomicroscope on unfixed tissues. Hind leg muscle was used as anegative control. For flow cytometric analysis the teratoma was cut intosmall pieces using a scalpel blade and subsequent treatment withCollagenase B for 45 m at 370 C. Cells were then passed several timesthrough a 21 G needle and subsequently treated with Trypsin-EDTA for 5m. After 2 washes the cells were analyzed on an LSRII flow cytometer.Dead cells were excluded using forward and side scatter parameters. Forhistological analysis tissue was embedded into paraffin blocks and thesections stained with hematoxylin/eosin (H&E).

Example 1 Identification of hRosa26

Using the sequence of mouse Rosa26 transcripts (1) as a template tosearch the Ensembl database (3), a region with several highlysignificant homologies on human chromosome 3 was located. The highestdegree of sequence similarity (>85%) was found in a stretch ofnon-repetitive DNA corresponding to the 5′ portion of exon 1 plus ˜1.0kb of the putative promoter region in the mouse Rosa26 locus (FIG. 5a ).An electronic screen of the Ensembl gene expression database for thisgenomic region revealed a large number of uncharacterized expressedsequence tags (ESTs) and transcripts. These transcripts are derived froma broad spectrum of adult cell types and 3 embryonic stem cell lines.They map to specific positions within the region of Rosa26-synteny, mostlikely demarcating exons (FIG. 5b ). Thus, the region on humanchromosome 3 identified represents the equivalent of the mouse Rosa26locus and should thus be referred to as human Rosa26 (hRosa26).

To begin to define the expression pattern of the hRosa26 locus differentadult tissues were analyzed for hRosa26 message by quantitativepolymerase chain reaction (qPCR) using oligonucleotides against thesequences in putative ExonIII As shown in FIG. 1a , hRosa26 wasexpressed in all human tissues tested as well as in 3 different hESClines. The levels of expression in adult human tissue varied between˜0.2× and ˜1.4× of the levels detected in H1 hESCs. Comparableexpression patterns were observed using oligonucleotides that span thepredicted intron between ExonII and ExonIII in a conventional RTPCRreaction (FIG. 1b ). The different levels of expression observed inthese human tissues are not unexpected as the mouse Rosa26 locus alsoshows variable expression in adult tissues.

Example 2 Construction of a hRosa26 Targeting Vector

A targeting vector analogous to the widely used mouse Rosa26 vector (2)was constructed (FIG. 1c ). The vector contains a 5′ short arm and a 3′long arm of homology, which together span approximately 5.1 kb of thehRosa26 locus. The vector overlaps with sequences of the putative firstexon and the following intron of the hRosa26 locus. The homologous armsare separated by an expression cassette which consists of severalelements, including a viral splice acceptor, followed by a loxP-flankedpromoterless neomycin resistance gene. The neomycin gene is followed byan inverted RFP variant (5), termed tandem-dimer RFP (tdRFP), flanked bymutant loxP2272 sites (6). tdRFP is a covalently fused dimer of thegenetically modified monomeric RFP. This was selected because it maturesfast (about 2 hours) (7), is not toxic in mouse ES cells (8) and issignificantly brighter than monomeric RFP variants such as mRFP (1) ormCherry, thus facilitating transgene detection during in vitrodifferentiation and teratoma formation. The loxP and mutant loxP2272sites are positioned such that following expression of Cre recombinase(Cre) (9, 10), the neomycin resistance cassette is removed and the tdRFPinverted, placing it under control of the endogenous hRosa26 promoter(FIG. 1d ).

Two distinct intermediates can be generated during this process, asrecently demonstrated by Luche et al (8). This gene trap construct willfunction as a Cre inducible fluorescent reporter after successfultargeting of the locus. By using an inverted tdRFP cassette rather thana “foxed” stopper cassette (11), the possibility of low levels of“leaky” tdRFP expression detected in previous studies using a similarconstruct in mouse ES cells was eliminated (8). Furthermore the use ofheterotypic loxP sites allows for the subsequent exchange of the tdRFPcassette with virtually any cDNA of interest at the hRosa26 locus usingrecombinase mediated cassette exchange (RMCE)(12).

Example 3 Gene Targeting

For gene targeting, the hESC line HES213 was electroporated with thelinearized targeting construct. One day following electroporation,selection with neomycin sulfate (G418) was initiated. G418 resistantclones, detected 10-12 days later, were picked and analyzed by PCR forevidence of correct targeting using a mock plasmid with an extended 5′homology region as a positive control. Out of 24 clones analyzed one wascorrectly targeted based on this PCR analysis. This clone was expandedand electroporated with a plasmid that expressed Cre transiently.Forty-eight hours later, tdRFP positive cells, which comprised about 5%of the total population, were isolated by cell sorting and cultured atlow density. Colonies expressing tdRFP, identified by fluorescencemicroscopy, were picked and expanded. Correctly targeted clones wereconfirmed by Southern blot analysis using an external genomic probe(FIG. 1e ). The integration of a single copy of the targeting constructwas confirmed by the appearance of a single band of the expected sizeusing a probe specific for the tdRFP cassette (FIG. 1f and FIG. 5).Cre/loxP-mediated deletion of the neomycin cassette and maintenance ofthe targeted locus was confirmed by loss of G418 resistance and Southernblot analysis (FIG. 1e ). One human hRosa26 tdRFP subclone (hRosa26 B72.12) was used for most of the subsequent analysis. This clone will bereferred to as hRosa26.

From this first set of experiments screening of 40 more clones bySouthern blotting revealed no additional, correctly targeted clones(Maximum targeting efficiency 1 in 64, ˜1.5%). In a second experiment,one targeted clone was identified (Southern blot) out of 24 clonestested (1 in 24, ˜4.2%. overall 2 in 88, ˜2.3%). This clone is referredto as hRosa26.2 (FIG. 6a ). While the 5 observed targeting frequenciesare lower than reported for the mouse Rosa26 locus2, they appear to bein a range well acceptable for multiple targeting experiments. Whetherthe lower targeting frequency is due to locus-specific speciesdifferences with regard to homologous recombination or to more trivial,technical issues, such as suboptimal DNA delivery and selectionmechanisms, needs to be investigated in a systematic manner. With onlyone previous report of gene targeting in hESCs (14) critical parametersfor optimization of homologous recombination in human ES cells remainsto be determined Chromosomal analysis of the targeted clone following 8passages after the second electroporation confirmed a normal karyotype(46XX). The undifferentiated cells displayed a typical ES cellmorphology (FIG. 2a ), expressed significant levels of tdRFP, readilydetectable by fluorescent microscopy (FIG. 2b ) and flow cytometricanalysis (FIG. 2f ). These colonies were positive for alkalinephosphatase (FIG. 2c ). When induced to differentiate in serum freemedia, the hRosa26 tdRFP cells formed embryoid bodies (EBs, FIG. 2d ).tdRFP expression could be demonstrated by epifluorescent microscopy (day4 EBs, FIG. 2e ) and flow cytometric analysis at day 17 ofdifferentiation (FIG. 2f ). Eight to ten weeks following injection intothe hindlimb of NOD/SCID mice, hRosa26 hESCs generated teratomas (FIG.2g ), that grossly expressed tdRFP (FIG. 2h ). Immunofluoresecent(insert FIG. 2h ) and flow cytometric analysis of single cells derivedfrom a second teratoma generated from another hRosa26 subclone (B7 2.10)revealed that most cells (>96%) expressed tdRFP (FIG. 2i ).

The small population (<4%) of tdRFP-cells may represent the vasculatureof the host. One of the hallmarks of ES cells is their capacity todifferentiate to multiple cell types representing derivatives of the 3germ layers. FIG. 7 shows histological analysis of a hRosa26hESC-derived teratoma revealing the presence of cartilage and cardiacmuscle (mesoderm), ciliated mucosal tissue (endoderm) and neuralrosettes (ectoderm).

Example 4 Cell Differentiation Experiments

To monitor tdRFP expression in differentiated progeny generated invitro, the targeted hESCs were induced to differentiate into cellsrepresenting derivatives of the 3 embryonic germ layers ectoderm,endoderm and mesoderm as well as the trophoblast lineage, anextraembryonic population.

Example 4a Ectoderm Expression

To evaluate expression of tdRFP in ectoderm, both wild type and hRosa26ES cells were induced to differentiate to the neuronal lineage. For thisdifferentiation, the hESCs were removed from the stem cell conditionsand differentiated as EBs in serum free media. Basic fibroblast growthfactor (bFGF) was added to the EB cultures at day 4 of differentiationand at day 6 the EBs were transferred to gelatin coated 6-well-platesand cultured for an additional 21 days in serum-free media supplementedwith epidermal growth factor (EGF). These conditions supported thedevelopment of cells that express the neuronal marker β-Tubulin III15.These β-Tubulin III+ neuronal cells retained tdRFP expression (FIG. 3a ,insert) demonstrating that the human Rosa26 locus remains activefollowing differentiation of hESCs to neuroectoderm. The HES2 parentalcell line generated β-Tubulin III+ neurons that did not exhibit redfluorescence. Neuronal cells generated from both the hRosa26 and wildtype cells also expressed the neural cell adhesion molecule (NCAM).

Example 4b Endoderm Expression

It has been shown that activin A can induce definitive endoderm frommouse ES cells in the absence of serum (16). D'Amour et al. recentlyshowed that this factor also functions to induce endoderm in hESCscultures (17, 18). To test the endoderm potential of the hRosa26targeted hESCs they were differentiated as EBs in serum free conditionsin the presence of activin A. Following 8 days of induction the EBs weretransferred to gelatin coated 6-well plates in serum free mediumcontaining vascular endothelial growth factor (VEGF), basic fibroblastgrowth factor (bFGF) and bone morphogenic protein 4 (BMP4). Thesefactors have been shown to be important for liver specification in themouse embryo (19) and mouse embryonic stem cell differentiation cultures(20). Clusters of albumin and alpha-fetoprotein (AFP) positive cellscould be detected following 16 days of culture. As shown in FIG. 3b ,the AFP-expressing cells generated from the Rosa26 targeted hESCsexhibited bright red fluorescence whereas those from the parental cellline did not. These findings indicate that the human Rosa26 locus isalso expressed in endoderm derived cell populations.

Example 4c Mesoderm Expression

To evaluate expression in mesoderm derivatives, the Rosa26 targetedhESCs were differentiated to the hematopoietic lineage by induction withBMP4 in serum free medium (21, 22). At day 18, EBs were dissociated andreplated into methylcellulose containing a broad spectrum ofhematopoietic cytokines, conditions that will support the development ofboth erythroid and myeloid hematopoietic colonies. As shown in FIG. 3c ,both types of colonies expressed tdRFP. CD45, a pan specifichematopoietic marker, is expressed on hematopoietic cells at days 14 and21 of EB differentiation (FIG. 3d ). At these time points both CD45− andCD45+ cells released into the culture medium expressed tdRFP. Theparental wild type hESCs derived cells were tdRFP negative. The secondtargeted cell line, hRosa26.2 also generated CD45+ cells that expresshigh levels of tdRFP following induction with BMP4 (FIG. 6b ).

Example 4d Expression of tdRFP in an Extraembryonic Population

To test the expression of tdRFP in an extraembryonic population, wildtype and hRosa26 hESC were induced to differentiate to the trophoblastlineage by treatment with high levels of human recombinant BMP423. Asshown in FIG. 3e derivatives of both the parental HES2 (right) and thetargeted hRosa26 (left) hESCs were able generate cells that secret humanchorionic gonadotropin. Only those generated from the targeted hESC lineexpressed tdRFP. Taken together, these findings clearly demonstrate thatthe hRosa26 locus is expressed in derivatives of all three germ layersas well as in trophoblast cells generated in culture.

Example 5 RMCE

The targeted insertion of loxP sites along with tdRFP introduced ahoming site for subsequent RMCE—a method that would allow the insertionof any sequence of interest into the hRosa26 locus without the need ofgene targeting or drug selection. To test the feasibility of thisapproach in hESCs, an exchange vector was engineered containing apromoterless puromycin resistance cassette (puro) flanked by a 5′ loxPand a 3′ loxP2272 site (FIG. 8b ). This exchange vector, together with aCre expression plasmid was electroporated into one hRosa26 clone. Fortyeight hours later, tdRFP negative cells (those that lost the tdRFPinsert) were isolated by cell sorting and cultured on MEF cells in theabsence of any drug selection. Approximately 20 clones grew from 10.000tdRFP-sorted cells within 14 day of culture. Four of these 20 cloneswere tdRFP negative as determined by epifluorescent microscopy and ofthese, 2 were resistant to puromycin. It is unclear why the majority ofthe clones generated from the tdRFP-cells consistently regained tdRFPexpression. One possibility is that the tdRFP coding sequencere-inserted into the site following Cre excision, and in doing so,established the original confirmation of the locus. The physicalorganization of the targeted locus was analyzed in the 4 tdRFP negativeclones (hRosa26_puro#1-4). Southern blot analysis revealed that the 2puromycin resistant clones (hRosa26_puro#1+4) each contained thepredicted band indicative of a single integration of the puromycincassette following hybridization with a puro specific internal probe(FIG. 8d ). Both the wild type and targeted alleles were detectedfollowing hybridization with an external probe (FIG. 8e ). The 2tdRFP-clones (hRosa26_puro#2+3) that were sensitive to puromycin lostthe targeted allele, presumably due to homologous recombination mediatedrepair by the sister chromosome after the Cre induced double strandbreak. Neither of these clones contained a detectable puromycin specificband in a Southern blot analysis (FIG. 8d ). This exchange in the twopuromycin-positive clones was not driven by a selection withantibiotics, but rather was dependant on the loss of tdRFP, illustratingthe potential of this method as a universal strategy to exchange tdRFPwith otherwise non-selectable genes. Subsequently, two additionalexchange vectors were designed, one expressing EGFP and the otherLHX224. Preliminary analysis with these vectors indicate an exchangefrequency of 1 in 9 and 3 in 5 of the tdRFP-clones, respectively.Improving DNA delivery conditions and varying the ratio of exchangeplasmid to Cre expression plasmid may further improve the favoredoutcome. Together these findings clearly demonstrate that it is possibleto introduce cDNA's of interest into the hRosa26 lcous of the Rosa26hESC through RMCE.

REFERENCES

-   1. Zambrowicz, B. P. et al. Disruption of overlapping transcripts in    the ROSA beta geo 26 gene trap strain leads to widespread expression    of betagalactosidase in mouse embryos and hematopoietic cells. Proc    Natl Acad Sci USA 94, 3789-94 (1997).-   2. Soriano, P. Generalized lacZ expression with the ROSA26 Cre    reporter strain. Nat Genet 21, 70-1 (1999).-   3. Birney, E. et al. Ensembl 2006. Nucleic Acids Res 34, D556-61    (2006).-   4. Friedrich, G. & Soriano, P. Promoter traps in embryonic stem    cells: a genetic screen to identify and mutate developmental genes    in mice. Genes Dev 5, 1513-23 (1991).-   5. Campbell, R. E. et al. A monomeric red fluorescent protein.    Proceedings of the National Academy of Sciences of the United States    of America 99, 7877 (2002).-   6. Siegel, R. W., Jain, R. & Bradbury, A. Using an in vivo phagemid    system to identify non-compatible loxP sequences. FEBS Lett 499,    147-53 (2001).-   7. Shaner, N. C. et al Improved monomeric red, orange and yellow    fluorescent proteins derived from Discosoma sp. red fluorescent    protein. Nat Biotechnol 22, 1567-72 (2004).-   8. Luche, H., Weber, O., Rao, T. R., Blum, C. & Fehling, H. J.    Faithful activation of an extra-bright red fluorescent protein in    “knock-in” Cre-reporter mice ideally suited for lineage tracing    studies. Eur J Immunol 37, in press (2007).-   9. Schnutgen, F. et al. A directional strategy for monitoring    Cre-mediated recombination at the cellular level in the mouse. Nat    Biotechnol 21, 562-5 (2003).-   10. Sternberg, N. & Hamilton, D. Bacteriophage P1 site-specific    recombination. I. Recombination between loxP sites. J Mol Biol 150,    467-86 (1981).-   11. Mao, X., Fujiwara, Y., Chapdelaine, A., Yang, H. & Orkin, S. H.    Activation of EGFP expression by Cre-mediated excision in a new    ROSA26 reporter mouse strain. Blood 97, 324-6 (2001).-   12. Bouhassira, E. E., Westerman, K. & Leboulch, P. Transcriptional    behavior of LCR enhancer elements integrated at the same chromosomal    locus by recombinase-mediated cassette exchange. Blood 90, 3332-44    (1997).-   13. Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A. &    Bongso, A. Embryonic stem cell lines from human blastocysts: somatic    differentiation in vitro. Nat Biotechnol 18, 399-404 (2000).-   14. Zwaka, T. P. & Thomson, J. A. Homologous recombination in human    embryonic stem cells. Nat Biotechnol 21, 319-21 (2003).-   15. Reubinoff, B. E. et al. Neural progenitors from human embryonic    stem cells. Nat Biotechnol 19, 1134-40 (2001).-   16. Kubo, A. et al. Development of definitive endoderm from    embryonic stem cells in culture. Development 131, 1651-62 (2004).-   17. D'Amour, K. A. et al. Efficient differentiation of human    embryonic stem cells to definitive endoderm. Nat Biotechnol 23,    1534-41 (2005).-   18. D'Amour, K. A. et al. Production of pancreatic    hormone-expressing 22 endocrine cells from human embryonic stem    cells. Nat Biotechnol 24, 1392-401 (2006).-   19. Rossi, J. M., Dunn, N. R., Hogan, B. L. & Zaret, K. S. Distinct    mesodermal signals, including BMPs from the septum transversum    mesenchyme, are required in combination for hepatogenesis from the    endoderm. Genes Dev 15, 1998-2009 (2001).-   20. Gouon-Evans, V. et al. BMP-4 is required for hepatic    specification of mouse embryonic stem cell-derived definitive    endoderm. Nat Biotechnol 24, 1402-11 (2006).-   21. Johansson, B. M. & Wiles, M. V. Evidence for involvement of    activin A and bone morphogenetic protein 4 in mammalian mesoderm and    hematopoietic development. Mol Cell Biol 15, 141-51 (1995).-   22. Kennedy, M., D'Souza, S. L., Lynch-Kattman, M., Schwantz, S. &    Keller, G. Development of the hemangioblast defines the onset of    hematopoiesis in human ES cell differentiation cultures. Blood    (2006).-   23. Xu, R. H. et al. BMP4 initiates human embryonic stem cell    differentiation to trophoblast. Nat Biotechnol 20, 1261-4 (2002).-   24. Xu, Y. et al. LH-2: a LIM/homeodomain gene expressed in    developing lymphocytes and neural cells. Proc Natl Acad Sci USA 90,    227-31 (1993).-   25. Costa, M. et al. The hESC line Envy expresses high levels of GFP    in all differentiated progeny. Nat Methods 2, 259-60 (2005).-   26. Nolden, L. et al. Site-specific recombination in human embryonic    stem cells induced by cell-permeant Cre recombinase. Nat Methods 3,    461-7 (2006).-   27. Gerrard, L., Zhao, D., Clark, A. J. & Cui, W. Stably transfected    human embryonic stem cell clones express OCT4-specific green    fluorescent protein and maintain self-renewal and pluripotency. Stem    Cells 23, 124-33 (2005).-   28. Muotri, A. R., Nakashima, K., Toni, N., Sandler, V. M. &    Gage, F. H. Development of functional human embryonic stem    cell-derived neurons in mouse brain. Proc Natl Acad Sci USA 102,    18644-8 (2005).-   29. Thomson, J. A. et al. Embryonic stem cell lines derived from    human blastocysts. Science 282, 1145-7 (1998).-   30. Kennedy, M. & Keller, G. M. Hematopoietic commitment of ES cells    in culture. Methods Enzymol 365, 39-59 (2003).

We claim:
 1. A method for modifying a human Rosa26 gene in an isolatedhuman stem cell, which method comprises introducing an expressioncassette comprising a nucleic acid sequence encoding a protein, suchthat said nucleic acid sequence is operatively linked to a promoterheterologous to the human Rosa26 gene, or the endogenous human Rosa26promoter.
 2. The method of claim 1, wherein the expression cassette isintroduced into the human stem cell by homologous recombination with atargeting vector comprising said expression cassette flanked by DNAsequences homologous to the human Rosa26 gene.
 3. The method of claim 1,wherein the human stem cell is a primary cell or an immortalized cell.4. The method of claim 1, wherein the nucleic acid sequence encodes arecombinase, or a reporter.
 5. The method of claim 1, wherein thepromoter heterologous to the human Rosa26 gene is selected from thegroup consisting of a constitutive ubiquitous promoter, a constitutivetissue specific promoter, an inducible ubiquitous promoter, and aninducible tissue specific promoter.
 6. The method of claim 1 wherein theexpression cassette further comprises a marker gene, one or morerecombinase recognition site, a poly A signal, an intron, orcombinations thereof.
 7. The method of claim 1, wherein the expressioncassette comprises a viral splice acceptor, a loxP-flanked promoterlessneomycin resistance gene, an inverted RFP variant, loxP2272 sites, orcombinations thereof.
 8. The method of claim 1, wherein the expressioncassette comprises the following elements in sequential order: (a) aviral splice acceptor, (b) a loxP site, (c) a promoterless neomycinresistance gene, (d) a loxP2272 site, (e) an inverted nucleic acidsequence encoding the protein, (f) a loxP site, and (g) a loxP2272 site.9. The method of claim 2, wherein the targeting vector further comprisestags for protein detection, enhancers, selection markers, orcombinations thereof.
 10. The method of claim 1, wherein said promoteris heterologous to the human Rosa26 gene.
 11. The method of claim 1wherein said promoter is the endogenous Rosa26 promoter.